The efficient removal of impurities during affinity chromatography, including host cell proteins (HCPs) and product-related impurities, such as high (HMWs) and low (LMWs) molecular weight species, is a crucial factor during downstream processing of proteins. The purity of the protein after the first purification step (the “capture step”), notably influences the type and number of subsequent steps required to generate a purified product. The capture step is also critical because it concentrates the product, which allows for the use of proportionally smaller, less costly columns in subsequent purification steps. Therefore, it is important to optimize the removal of impurities during the first chromatography step. In the case of antibody purification, this first step is typically based on affinity to protein A or derivatives.
Low pH conditions (e.g., between pH 3-4) are required to elute bound target protein from the affinity column, but have the disadvantage of potentially inducing aggregation. Historically, less stringent conditions, such as a pH between 5-5.5 have been used to wash non-specifically bound impurities from the column, while simultaneously preserving the target-protein A interaction. The recovery, however, is often decreased due to partial elution of the target protein under these conditions, particularly when working at high loading densities.
The amino acid arginine has been shown to solubilize certain precipitated proteins (M, Tsumoto K, Nitta S, Adschiri T, Ejima D, Arakawa T, and Kumagai I. Biochem. Biophys. Res. Commun. 328, 189-197 (2005); Umetsu M, Tsumoto K, Hara M, Ashish K, Goda S, Adschiri T, Kumagai I. J Biol Chem. 2003 Mar. 14; 278(11):8979-87), reduce the formation of aggregates (Arakawa T, Tsumoto K. Biochem Biophys Res Commun. 2003 Apr. 25; 304(1):148-52) and Arakawa T, Biophys. Chem. 127 (2007), pp. 1-8 (Review)) and reduce nonspecific adsorption of proteins to surfaces (Ejima D, J Chromato A, 05 and Schneider C P, J. Phys. Chem. B 113 (2009), pp. 2050-2058). Moreover, in contrast to guanidium hydrochloride, arginine has not been shown not to unfold proteins (Arakawa T, Biochem. Biophys. Res. Commun. 304 (2003), pp. 148-152 and Nakakido M, Biophys. Chem. 137 (2008), pp. 105-109).
As such, arginine has been used to elute proteins from affinity chromatography columns and other types of purification columns. For example, Arakawa et al. describe methods of eluting antibodies from a Protein A column using an elution buffer containing 0.5-2.0 M arginine at pH 4.1-5.0 (Arakawa et al. (2004) Protein Expression and Purification 36:244-248; Tsumoto, K. et al. (2004) Biotechnol. Prog. 20:1301-1308; U.S. Patent Publication No. 20050176109). Additionally, U.S. Pat. No. 7,501,495 by Ejima et al. describes methods of eluting proteins from a gel filtration column by a developing solution containing arginine hydrochloride. Ghose et al. describe methods of eluting proteins of interest from underivatized silica using an arginine gradient as the eluant (Ghose, S. et al. (2004) Biotech. Bioeng. 87:413-423). U.S. Patent Publication No. 20030050450 by Coffman et al. describes methods of dissociating Fc containing molecules from complexes of the Fc containing molecule and Protein A, wherein the Fc/Protein A complexes are applied to a hydrophobic interaction column (HIC) and the column is washed with a buffer containing arginine.
Barron et al. describe an intermediate wash solution for Protein A chromatography containing 0.5 to 2.0 M arginine in a phosphate/acetate buffer at pH 5.0-7.5 (optimally 1M arginine, 0.1M phosphate/acetate buffer at pH 5.0). This arginine wash step is reported to remove HCP contaminants. The authors also tested an intermediate wash solution that contained sodium chloride at 0.5-2.0 M at pH 5.0-7.5, and reported that the NaCl wash showed no significant decrease in HCP (Barron et al., “Improving Purity on Protein A Affinity Media Through Use of an Arginine Intermediate Wash Step”, http://www.priorartdatabase.com/IPCOM/000127319). Moreover, Barron et al. reported that lowering the pH of the wash buffer had a beneficial effect under the conditions used in the experiment.
There is a longstanding need for improved techniques to enhance the purification process and increase product recovery. The present disclosure addresses this need and provides additional benefits.